小干扰RNA对肝星状细胞基质金属蛋白酶组织抑制因子-1表达的抑制作用

目的观察化学合成的小干扰RNA(siRNA)转染大鼠肝星状细胞(HSC)-T6后不同时间点,对基质金属蛋白酶组织抑制因子-1(TIMP-1)的抑制作用,同时筛选高效的siRNA片断。方法对大鼠TIMP-1 mRNA nt161~181、nt 190~208、nt 208~226、nt 226~244及nt 445~463化学合成的5对siRNA和1对荧光标记的非特异siRNA(与TIMP-1 mRNA无同源性的FITC标记的21nt siRNA),在阳离子脂质体的介导下将不同浓度的非特异siRNA转染至大鼠HSC-T6后,用流式细胞仪确定最佳转染浓度。提取转染50 nmol/L siRNAs后24 h,48 h和72 h细胞蛋白,用W estern b lot检测TIMP-1蛋白质表达,筛选出抑制效率最高的siRNA,同时确定siRNA转染细胞后的最佳作用时间。结果50 nmol/L siRNAs对HSC-T6有较高的转染效率;5对siRNA中的3对在转染后48 h有较强的抑制HSC-T6细胞TIMP-1基因表达的作用,其中抑制作用最强的1对siRNA对TIMP-1表达的抑制作用与对照组相比可增强80%以上,而在转染后72 h,其抑制作用基本消失。结论化学合成的siRNA在短时期内可有效地抑制TIMP-1基因的表达,筛选到的高效阻抑TIMP-1表达的siRNA可构建于病毒载体,以实现长期抑制TIMP-1表达的功效。

Suppression of Tissue Inhibitor of Metalloproteinase-1 on Gene Expression by Small Interfering RNA in Rat Hepatic Stellate Cell

Objective To investigate the effects of RNA interference targeting TIMP-1 gene on rat hepatic stellate cell(HSC)-T6 cells in vitro and screen highly efficient small interfering RNA(siRNA).Methods Five pair of 21 nucleotide siRNAs targeting rat TIMP-1 at nt161~181,nt 190~208,nt 208~226,nt 226~244,nt 445~463 and one pair of fluorescence labeled unspecific siRNA were synthesized.After transfecting different concentrations of unspecific siRNA to select the optimum transfection concentration,50 nmol/L siRNAs were transfected into rat HSC-T6 cells respectively.The protein of the infected cells at 24 h,48 h and 72 h was collected and detected by Western blot to confirm the suppression effects of diverse siRNAs at different time point.Results It was demonstrated by fluorescence actived cell sorter that 50 nmol/L siRNA had the optimum trasfection efficacy,and 3 of the 5 siRNA oligomers had the significant effects on suppressing TIMP-1 expression by Western-blot within 48 hours after transfecting into rat HSC-T6 cells.The expression level of TIMP-1 gene in HSC-T6 cells infected by one pair of siRNA after 48 h decreased significantly(80%) compared with control cells,and the effects of suppression disappeared at 72 h after transfection.Conclusion RNA interference can exert an suppression of TIMP-1 gene in rat HSC.One paie of siRNA,which can highly effectively inhibit expression of TIMP-1 gene was screened successfully,and it will be reconstructed into virus vectors to suppress the specific gene expression for a long time by chromosomal integration.